A Mixture of α-Helical and 310-Helical Conformations for Involucrin in the Human Epidermal Corneocyte Envelope Provides a Scaffold for the Attachment of Both Lipids and Proteins*

Involucrin plays an important role in the lipid and protein compound envelopes of mammalian epidermal corneocytes. In the present study, model peptides containing the consensus repeating units PEQQEGQLEL and LEQQEGQLEH, found in the central region of human involucrin, were studied by circular dichroism spectroscopy, molecular modeling, and energy minimization. These peptides have intrinsic α-helix-forming properties as indicated by their circular dichroic spectra obtained in the presence of 2,2,2-trifluoroethanol. Peptide (LEQQEGQLEH)3 had an α-helix content of 100% in 100% 2,2,2-trifluoroethanol at 0 °C. The energy-minimized α-helix showed that only 50% of the glutamate side chains may be available for the attachment of lipids. However, when a 310-helix was assumed for the GQL or GQLE residues in LEQQEGQLEH, all of the glutamate side chains were arrayed on one face of the helix, and all of the glutamine side chains were arrayed on the opposite face. A similar result was obtained when the nonhelical part of PEQQEGQLEL was assumed to contain a β-turn III, which is equivalent to a short portion of 310-helix. The results of this study suggest that when the central segment of human involucrin is predominantly α-helical, accompanied by short 310-helical segments, the protein can function as a scaffold for the attachment of both lipids and proteins.

Corneocytes are the thin, flat cells that form the outer surface of mammalian skin, and they play a vital role in the epidermal permeability barrier. Each corneocyte possesses a protein envelope, approximately 15 nm in thickness, which is composed of several structural proteins including involucrin (1)(2)(3)(4), cystatin ␣ (5, 6), loricrin (7)(8)(9), elafin (10), small prolinerich proteins (11,12), keratin intermediate filaments (13), trichohyalin (14), and filaggrin (15). These proteins are crosslinked together by N ⑀ -(␥-glutamyl)lysine isodipeptide bonds formed by the action of transglutaminases and also by disulfide bonds. Studies of the structure and assembly of the protein envelope indicate that involucrin is one of the proteins that is deposited first and that it forms a scaffold for the subsequent deposition of the other proteins (3, 16 -18).
Covalently attached to the exterior of the corneocyte protein envelope is a lipid envelope composed mainly of -hydroxycer-amides (19,20). Chemical evidence indicates that these lipid molecules are attached to proteins through ester bonds, since they are easily released by treatment with mild alkali (19,20). The availability of sufficient ester bonds requires a protein that is rich in free carboxyl side chains (21). Thus, involucrin, which has a high glutamate content (Ϸ20% of the amino acids), has been proposed to act as a scaffold for the attachment of the lipid layer to the protein envelope (22). This idea was confirmed by biochemical and mass spectrometric characterization of lipopeptide fragments obtained from human corneocyte envelopes by proteolysis, which indicated that approximately 35% of the peptides were derived from involucrin (23). Thus, from studies of protein and lipid envelopes, it is generally accepted that involucrin is covalently attached to both proteins and lipids.
Biophysical characterization of human involucrin using secondary structure prediction, CD 1 and electron microscopy indicated that involucrin is a rod-shaped molecule that possesses 50 -75% ␣-helical content (4). It was suggested that the glutamine residues are circumferentially distributed along the length of the ␣-helix and that this arrangement maximizes intermolecular cross-linking between involucrin and other proteins (4). The distribution of glutamate residues and its role in the attachment of lipids were not addressed in that study. Initially, we predicted that human involucrin might adopt a ␤-sheet structure, since this could provide the required surface density of glutamate residues for the attachment of lipids (22). This prediction seemed to be supported by solid-state nuclear magnetic resonance studies of corneocyte envelopes isolated from pig epidermis, which suggested that the cross-linked proteins are predominantly in the ␤-sheet conformation (24). X-ray diffraction studies of murine corneocyte envelopes also indicated the dominant presence of ␤-sheets (25). However, human involucrin is a distinctly different protein than that of lower mammals (26), and therefore, care must be taken in extrapolating results with lower mammals to humans.
In the present study, we used model peptides to show that the ␣-helical character that has been demonstrated for the involucrin molecule as a whole (4) can be adopted by the specific regions that are rich in glutamate residues. Also, using computer modeling, we investigated whether an ␣-helical conformation would array the glutamate residues of these regions appropriately for attachment of the lipid envelope. Our results suggest that a mixture of ␣-helical and 3 10

EXPERIMENTAL PROCEDURES
Peptide Synthesis and Characterization-Peptides were synthesized in the Protein Structure Facility of the University of Iowa using an Applied Biosystems 431A peptide synthesizer. A stepwise solid-phase procedure using rink amide MBHA resin and fluorenylmethoxycarbonyl-protected amino acids was employed. The crude peptides were purified by preparative C18 reverse-phase HPLC. The purity of all peptides used in this study was confirmed to be Ͼ95% by analytical C18 reverse-phase HPLC. Peptide identity was confirmed by electrosprayionization mass spectrometry and by amino acid analysis.
Circular Dichroism-CD spectra were acquired using an Aviv Associates model 62DS spectrometer. Stoppered optical cells with a path length of 1 mm were used in all acquisitions. Spectra were obtained by taking readings every 0.5 nm with an averaging time of 4 s and a bandwidth of 1 nm. Ellipticity measurements were expressed as mean residue ellipticity, , in units of deg cm 2 dmol Ϫ1 and calculated from the equation, in which is the observed ellipticity in degrees, MRW is the mean residue weight of the peptide (molecular weight divided by the number of residues), [C] is the peptide concentration in g/liter, and l is the optical path length in centimeters. Peptide concentrations were determined by quantitative amino acid amino acid analysis. The helix content of each peptide was determined from the values of their mean residue ellipticities at 222 nm ( 222 ). Based on CD studies of model peptides that mimic the ␣-helical regions of intermediate-filament proteins, the 222 for a 100% helix was taken as Ϫ43,000 deg cm 2 dmol Ϫ1 (27).
Computer Modeling-Energy minimization of possible structures for (PEQQEGQLEL) 1-3 and (LEQQEGQLEH) 1-3 was performed on a Silicon Graphics Indigo workstation in the Image Analysis Facility of the University of Iowa using Sybyl 6.4 software. The conformational energies were minimized using the Kollman all-atom force field and Kollman charges using 1,000 iterations or until convergence was reached. Energies for the 10-residue repeating units were calculated by subtracting the energy of the 20-residue peptide from that of the 30-residue peptide. These are reported here in units of kcal/mol-residue.

RESULTS
An important feature of human involucrin is the presence of a central segment of high homology whose consensus repeat sequence is (P/L)EQQEGQL(E/K)H (2). From CD, molecular modeling, and electron microscopy, it has been shown that the central segment contains a large proportion of ␣-helices (4). Our initial molecular modeling of this segment, using a graphical method developed for the expression of local spatial relationships between amino acid residues (28), is shown in Fig. 1. A predominantly ␣-helical conformation for the central segment is indicated, with most of the glutamate side chains arrayed on one face of the ␣-helices, and most of the glutamine side chains arrayed on the opposite face. To check the validity of this model, peptides containing the consensus 10-residue repeating units LEQQEGQLEH and PEQQEGQLEL were synthesized and investigated by CD spectroscopy, revealing random coil conformation in water ( Fig. 2) but ␣-helical structures in TFE (Fig.  3). In addition, (PEQQEGQLEL) 3 and (LEQQEGQLEH) 3 sequences were subjected to energy minimization by computer, confirming their predominantly ␣-helix-forming properties in conformations that would maximize the attachment of both lipids and proteins.
Model Peptides of Involucrin Possess Intrinsic Helix-forming Properties-CD spectra of the 20-and 30-residue peptides containing the repeating units PEQQEGQLEL and LEQQEGQLEH in phosphate-buffered saline at pH 7 show a strong minimum at approximately 198 nm, corresponding to the -* transition of the random coil (Fig. 2). Similar spectra were obtained after the addition of 2 molal sodium sulfate, a salt that is known to promote structure formation in peptides that contain hydrophobic residues. 2 At pH 2.7, below the pK a of the glutamate side chains, the peptides remained predominantly random coil.
␣-Helices were formed by all of the synthetic peptides in the presence of TFE, a nonpolar solvent that has been shown to promote native-like ␣-helical structures in peptides with intrinsic ␣-helix forming properties (29 -32). TFE has a dialectric 2 N. D. Lazo and D. T. Downing, unpublished observations. constant that closely approximates that of the interior of proteins and has recently been shown to promote structure formation by minimizing the exposure of the peptide backbone to water (30,32). Thus, solvents with high concentrations of TFE may mimic the hydrophobic environment of involucrin found in the corneocyte envelope. Fig. 3 presents CD spectra of (PEQQEGQLEL) 1-3 and (LEQQEGQLEH) 1-3 . Peptide (PEQ-QEGQLEL) 1 was insoluble in 100% TFE but was soluble in 25-75% TFE. The spectra of (LEQQEGQLEH) 2 and (LEQQEGQLEH) 3 in 75-100% TFE were characteristic of the dominant presence of an ␣-helix (33) (Fig. 3, e and f); each showed a minimum at 222 nm (n-* transition), a second minimum at 208 nm (-* transition polarized along the helix axis), and a maximum at 193 nm (-* transition polarized perpendicular to the helix axis). On the other hand, the spectra of (LEQQEGQLEH) 1 (Fig. 3d) and (PEQQEGQLEL) 1-3 (Fig. 3, a-c) had minima at approximately 220 nm and at 202-203 nm, indicating a lower proportion of ␣-helix. Table I shows the 222 and corresponding ␣-helix contents of the peptides in 100% TFE at 0°C. Peptides (PEQQEGQLEL) 2 and (PEQQEGQLEL) 3 had similar helix contents, which suggests that ␣-helix formation in these peptides is independent of chain length. In 25-75% TFE, the series of (PEQQEGQLEL) 1-3 peptides exhibited similar spectra, indicating similar helix contents (Fig. 3, a-c), perhaps because the ␣-helix formed by the PEQQEGQLEL repeating unit is interrupted by the proline of the next repeating unit. Proline lacks a peptide NH group that can participate in backbone hydrogen bonding, and because of its cyclic ring structure, it is energetically unfavorable for the preceding residue to form an ␣-helix (34). On the other hand, the helix content of peptides containing the LEQQEGQLEH repeating unit was dependent on the length of the chain (Fig. 3, d-f, Table I); peptide (LEQQEGQLEH) 3 had the highest helicity, with a 222 of Ϫ41,900 deg cm 2 dmol Ϫ1 , corresponding to an ␣-helix content of almost 100%.
An isodichroic point at 201.5 nm was observed for the (LEQQEGQLEH) 1-3 peptides (Fig. 3, d-f), which suggests that, under all conditions of TFE concentration and temperature used, each amino acid residue in the peptides existed as either random coil or ␣-helix (35). On the other hand, no isodichroic point was observed in the spectra of the (PEQQEGQLEL) 2 and (PEQQEGQLEL) 3 (Fig. 3, b-c), indicating the presence of more than two conformations.
Energy-minimized Structures Containing ␣-Helices with or without 3 10 -Helices-All residues of (LEQQEGQLEH) 3 were assigned backbone torsion angles corresponding to an ␣-helix ( ϭ Ϫ57°, ϭ Ϫ47°), and then the minimum conformational energy of the peptide was computed. Fig. 4a shows the ␣-helix after energy minimization. The conformational energy of the LEQQEGQLEH repeating unit was Ϫ16.3 kcal/mol-residue, which is lower than the energy of a corresponding ␤-strand (Ϫ14.0 kcal/mol-residue). However, in the pure ␣-helix, as shown in Fig. 4a, only 50% of the glutamate side chains may be available for the attachment of lipid. Therefore, possible modifications of the ␣-helical conformation of (LEQQEGQLEH) 3 that would maximize its potential for the attachment of both lipids and proteins were investigated. These modifications began with the glycine residue, which, primarily because of the conformational flexibility of its backbone (36), is a known ␣-helix breaker. It is also known that facile transitions between ␣-helix and 3 10 -helix occur in proteins and that these transitions play an important role in protein recognition and function (37)(38)(39). Most 3 10 -helices in proteins are short (3-4 residues long) compared with ␣-helices, which have a mean length of 10 residues (40). Fig. 4b shows the energy-minimized structure of (LEQQEGQLEH) 3 in which the segment GQL was modeled as a 3 10 -helix, i.e. the backbone torsion angles of GQL were initially set to ϭ Ϫ50°and ϭ Ϫ28°. In the resulting conformation, all of the glutamate side chains were arrayed on one face of the helix, and all of the glutamine side chains were arrayed on the opposite face. The computed conformational energy of the structure in Fig. 4b was Ϫ15.3 kcal/mol-residue.
In modeling (PEQQEGQLEL) 3 , it was initially assumed that the non-␣-helical part included proline and the preceding residue (34). To account for the 40% non-␣-helical content, two other residues next to LP were included as part of the non-␣helical segment, i.e. LPEQ, ELPE, and LELP were modeled as random coil. The resulting structures were energy-minimized, and their energies were compared. The lowest energy (Ϫ15 kcal/mol-residue) was obtained from the structure in which the LPEQ segment was assumed to be random coil. The energyminimized structure of (PEQQEGQLEL) 3 , composed of three ␣-helices separated from each other by the LPEQ random coil segments, was not linear. When the LPEQ segment was modeled as a type III ␤-turn, a structure showing all of the glutamate side chains on one face of the helix and all of the glutamine side chains on the opposite face, similar to Fig. 4, b and c, was obtained. The conformational energy of the structure was Ϫ15.9 kcal/mol-residue, which was lower than the energy of the structure in which LPEQ was modeled as random coil. DISCUSSION From independent studies of the lipid and the protein envelopes, it has been suggested that involucrin acts as a scaffold for the attachment of both proteins and lipids (3, 16 -22). For the protein to perform this function, it must present a surface that is rich in carboxylic acid groups opposite a surface that is rich in glutamines. In the present study we have shown that specific sequences constituting the central region of involucrin can adopt thermodynamically feasible ␣-helical plus 3 10 -helical conformations that fulfill the putative function of the protein, presenting all of the glutamate residues in a surface density adequate for attachment of the corneocyte lipid envelope.
The model peptides that we have investigated in this study represent two parts of the central segment of human involucrin. Residues 176 -225 of the protein are made up of five repeats of PEQQEGQLEL. Our studies of the peptides containing PEQQEGQLEL indicate that this segment of the protein may contain five ␣-helical regions separated from each other by four LPEQ segments. Energy calculations indicate that the LPEQ segments may form a ␤-turn III, equivalent to a segment of a 3 10 -helix (43), resulting in a linear structure in which all of the glutamate side chains are sequestered on one face, and all of the glutamine side chains are on the opposite face. The model peptides that contain the consensus repeat sequence LEQQEGQLEH possess intrinsic ␣-helix-forming properties that are dependent on chain length. This suggests that the sections that join the repeating units form part of the ␣-helix.
Previous molecular modeling of ␣-helices from the repeating unit LEQQEGQLKH by Yaffe and co-workers (4) show that when an extra amino acid is inserted between the consensus repeating units, a clustering of the glutamine residues on one face of the helix results. Here, we have shown that when an ␣-helix and a 3 10 -helix are present in the 10-residue repeating units, helical structures are obtained in which all of the glutamate side chains are sequestered on one face of the helix, and all of the glutamine side chains are clustered on the opposite face. The CD spectrum of human involucrin in phosphate buffer at neutral pH indicated a 222 of Ϫ18,000 deg cm 2 dmol Ϫ1 (4). Since a 3 10 -helix possesses an ␣-helix-like CD spectrum (41) and 3 10 -helices have been proposed as intermediates along the ␣-helix unfolding pathway (37,42), it has been proposed that a value between Ϫ15,000 and Ϫ30,000 deg cm 2 dmol Ϫ1 for the 222 of helical peptides or proteins suggests the presence of a mixture of nascent helix, 3 10 -helix, and ␣-helix (37).
Our energy calculations on the helical structures that contain the repeating unit LEQQEGQLEH indicate a slightly higher energy for the structures that contain 3 10 -helices than for the pure ␣-helix. The 3 10 -helix is more tightly wound and is less stable than the ␣-helix due to several close contacts and less optimal hydrogen bond geometry (44). However, there is no disallowed region between the ␣-helical and the 3 10 -helical conformations in the Ramachandran plot, and therefore ␣-helix to 3 10 -helix transformations can easily occur (44). Furthermore, calculations indicate that the low dielectric of proteins and the presence of lipid membranes could provide conditions that may stabilize the 3 10 -helix (45). In the corneocyte envelopes, the orienting effect of the bound lipids on one side of involucrin and the cross-linked proteins on the other side could also stabilize the 3 10 -helices. It also may be significant that esterification of the envelope lipids to the involucrin glutamates would remove the carboxylate electrostatic charges that might oppose ␣-helix formation. Furthermore, lateral hydrophobic interactions would tightly pack the attached envelope lipids, stabilizing and condensing the involucrin conformation.
There are two important reasons for having most if not all of the glutamate side chains sequestered on one face of involucrin in the human epidermal corneocyte envelope. First, it provides the high density of glutamate side chains required for attachment of the lipids in the interface between the protein and lipid compound envelopes of the corneocyte (19 -23). For this, every molecule in a tightly packed monolayer of hydroxyceramides is required to be attached through ester bonds to an underlying planar surface of protein (20 -22). Second, isolating the glutamate side chains on one face of involucrin may increase the FIG. 4. Energy-minimized structures of (LEQQEGQLEH) 3 . a, side view of a 100% ␣-helix; b, side view of ␣-helix ϩ 3 10 -helix at GQL; c, side view of ␣-helix ϩ 3 10 -helix at GQLE; and d, end views (from left to right) of structures shown in a, b, and c. The oxygens of the glutamate side chains (red) and nitrogens of the glutamine side chains (blue) are rendered as space-filling atoms. activity of transglutaminase for the formation of isodipeptide cross links, since it has been shown that in addition to being exposed on the surface of the protein, there should be no charged residues on either side of the target glutamine (46). The results presented here indicate that involucrin existing predominantly as ␣-helices and 3 10 -helices can function as a scaffold not only for the attachment of proteins but also for the attachment of lipids.